Dengue fever, dengue hemorrhagic fever, and dengue shock syndrome result from infection with one of four closely related serotypes of dengue virus, the most common vector-borne pathogen worldwide. These serotypes are designated dengue virus 1-4 (DEN 1-4), and they are transmitted by the mosquitoes Aedes aegypti and Aedes albopictus, which reside in tropical and sub-tropical areas of the world (Special Programme for Research and Training in Tropical Diseases, World Health Organization, (2009) Dengue: guidelines for diagnosis, treatment, prevention, and control. Geneva: TDR: World Health Organization). Infection with one serotype (primary infection) results in immunity to that serotype, but infection can occur with any of the remaining serotypes (secondary infection). Secondary infection has been shown to be a significant risk factor for the development of severe dengue, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) (Halstead et al. (1970) Yale J. Biol. Med. 42: 311-328). Recent reports estimate that 230 million infections occur annually, including 2 million cases of severe disease and 21,000 deaths. Over 3.6 billion people live in endemic regions and are at risk for infection (Gubler (2012) Am. J. Trop. Med. Hyg. 86: 743-744).
Despite the widespread nature of this disease, available diagnostic tests remain suboptimal (Special Programme for Research and Training in Tropical Diseases, World Health Organization, (2009) Dengue: guidelines for diagnosis, treatment, prevention, and control. Geneva: TDR: World Health Organization). The gold standard for diagnosis remains seroconversion, requiring collection of both acute and convalescent sera, and the gold standard for molecular diagnosis is a hemi-nested reverse transcription polymerase chain reaction (RT-PCR) assay originally developed in 1992 (Lanciotti et al. (1992) J. Clin. Microbiol. 30: 545-551). This assay requires two rounds of PCR followed by gel electrophoresis for amplicon detection. Not only do these numerous steps result in an increased risk of contamination, they also significantly limit the clinical utility of this assay, as the turnaround time is one day or longer. A number of other nucleic acid amplification tests have been developed for the detection and serotyping of dengue, including RT-PCR, real-time RT-PCR (rRT-PCR), and isothermal amplification techniques (Johnson et al. (2005) J. Clin. Microbiol. 43: 4977-4983; Kong (2006) J. Virol. Methods 138: 123-130; Hue et al. (2011) J. Virol. Methods 177: 168-173; and Munoz-Jordan et al. (2009) J. Clin. Microbiol. 47: 927-931). Many designs rely on two duplex reactions (two reactions per sample) or even four separate reactions for serotyping, and single-reaction, multiplex assays for detection and serotyping of dengue viruses lack the analytical sensitivity of the original hemi-nested RT-PCR (Johnson et al., supra; Hue et al., supra). Although these PCR assays are used around the world, an international external quality control assessment, published in 2010 involving 37 laboratories performing 46 tests, showed that 80% of these tests lacked sensitivity, specificity, or both (Domingo et al. (2010) PLoS Negl. Trop. Dis. 4(10) pii: e833).
Rapid and point-of-care assays based on the detection of IgM, IgG, and the non-structural 1 (NS1) protein have also been developed, but these all have significant limitations. IgM does not become detectable until at least the third day of clinical illness, but is not reliably positive until day five and beyond (Blacksell et al. (2006) Clin. Infect. Dis. 42: 1127-1134). These tests can be non-specific as IgM may persist two to three months following infection, and IgM against other flaviviruses cross-reacts with some tests (Blacksell et al. (2008) Diagn. Microbiol. Infect. Dis. 60: 43-49; Blacksell et al. (2011) Clin. Vaccine Immunol. 18: 2095-2101). IgG cannot distinguish current from past infection, an important issue in endemic areas where secondary infection is common. Assays based on detection of NS1 tend to be specific for dengue infection, but the sensitivity of these assays ranges widely in published reports from 24-93% (Guzman et al. (2010) PLoS Negl. Trop. Dis. 4; Tricou et al. (2010) BMC Infect. Dis. 10: 142). The sensitivity varies based on the specific assay used and the infecting serotype (Guzman et al., supra). All of the assays also show a roughly 20% decrease in sensitivity in the setting of secondary infection compared to primary infection (Tricou et al., supra; Chaterji et al. (2011) Am. J. Trop. Med. Hyg. 84: 224-228).
Thus, there remains a need for the development of effective strategies for the diagnosis, treatment, and prevention of dengue viral infection. The availability of nucleic acid diagnostic tests capable of efficiently detecting dengue virus in human specimens such as plasma, serum and respiratory secretions will assist the medical community in better diagnosing and treating dengue viral infections.